The present invention relates to transition metal complexes of N,Nxe2x80x2,Nxe2x80x3-trialkyl-cis,cis-1,3,5-triaminocyclohexane and related compositions and methods of synthesis and use in vitro and in vivo, such as a therapeutic agent or a delivery/imaging agent.
Nucleases that hydrolyze phosphate diester bonds exist in nature. Given that phosphate diester bonds are responsible for the accuracy of the genetic code, there has been interest in developing synthetic metallonucleases that hydrolyze phosphate diester bonds (Bashkin, Current Biology 7: R286-R288 (1997); Kuusela et al., Met. Ions Biol. Syst. 32: 271-300 (1996); Morrow, Met. Ions Biol. System. 33: 561-592 (1996); Sigman et al., Chem. Rev. 93: 2295-2361 (1993)). A variety of inert metal compounds have been evaluated as promoters (or catalysts) of activated phosphate diester hydrolysis (Williams et al., J. Am. Chem. Soc. 120: 8079-8087 (1998); Wahnon et al., Chem. Int. Ed. Eng. 34: 2412-2414 (1995); Hendry et al., In Progress in Inorganic Chemistry 38: 201-258, Lippard, ed., John Wiley and Sons, NY (1990); Hendry et al., Inorg. Chem 29: 92-97 (1990); Hendry et al., J. Am. Chem. Soc. 111: 2521-2527 (1989); Chin et al., J. Am. Chem. Soc. 111: 186-190 (1989)). Given that most natural nucleases contain one or two labile metal cations in the active site (Williams, In Comprehensive Biological Catalysts 1: 543-561, Sinnott, ed, Academic Press Ltd., San Diego, Calif. (1998); Cowan, Chem. Rev. 98: 1067-1087 (1998); Strater et al., Angew. Chem. Int. Ed. Engl. 35: 2024-2055 (1996)), recent efforts have focused on labile metal complexes like zinc and copper as synthetic nucleases (Yashiro et al., Chem. Commun. 1997: 83-84; Yashiro et al., J. Chem. Soc. Chem. Commun. 1995: 1793-1794; Gobel, Chem. Int. Ed. Engl. 33: 1141-1143 (1994); Koike et al., J. Am. Chem. Soc. 113: 8935-8941 (1991); de Rosch et al., Inorg. Chem. 29: 2409-2416 (1990); Morrow et al., Inorg. Chem. 27: 3387-3394 (1988)).
Copper (II) complexes have been explored. One of the first systems evaluated was Cu(2,2xe2x80x2-bipyridine)2+-catalyzed hydrolysis of ethyl(p-nitrophenyl)phosphate, and rate enhancements were reported (Morrow et al., Inorg. Chem. 27: 3387-3394 (1988)). Since then, others have explored bipyridine and pyridine derivatives as copper (II) ligands (Kovari et al., J. Am. Chem. Soc. 118: 12704-12709 (1996); Kovari et al., J. Chem. Soc. Chem. Commun. 1995: 1205-1206; Young et al., J. Am. Chem. Soc. 117: 9441-9447 (1995)). A variety of activated phosphate diesters and activated transesterification substrates, like bis(2,4-dinitrophenyl)phosphate (Young et al. (1995), supra), bis(p-nitrophenyl)phosphate (Kovari et al. (1996), supra; Kovari et al. (1995), supra), and 2-hydroxypropyl-p-nitrophenyl phosphate (hpnp; Wall et al., Angew. Chem. Int. Ed Engl. 32: 1633-1635 (1993); Wahnon et al., J. Chem. Soc. Chem. Commun. 1994: 1441-1442) have been used to determine the activity of the metal complexes.
Detailed kinetics and mechanistic studies of labile metal-promoted hydrolysis have been reported for the copper (II) triazacyclononane system (Deal et al., Inorg. Chem. 35: 2792-2798 (1996); see, also, Hegg et al., J. Am. Chem. Soc. 117: 7015-7016 (1995); Hegg et al., Inorg. Chem. 35: 7474-7481 (1996); and Hegg et al., Inorg. Chem. 36: 1715-1718 (1997)). Deal and co-workers measured a half-order metal complex dependence attributed to the formation of an inactive di-hydroxide bridged copper(II) dimer in equilibrium with the active mononuclear species (Deal et al. (1996), supra; Fromm, Initial Rate Enzyme Kinetics, Springer-Verlag, NY (1975), pp. 209-213). Studies by others with additional copper (II) complexes also have noted the half-order dependence (Wahnon et al. (1994), supra).
It has been suggested that a dinuclear copper complex which retains open coordination sites on the metal face may better promote phosphate diester hydrolysis (Deal et al., J. Am. Chem. Soc. 118: 1713-1718 (1996)). Wahnon and co-workers (Wahnon et al. (1994), supra) noted that the copper complex of bis(2-benzimidazolylmethyl)amine had an increase in the transesterification rate of hpnp with high metal complex concentrations, which was ascribed to an active dimer. The ligand is bulky, implying that steric constraints placed on the metal complex by the ligand may inhibit dimer formation.
Recently, Itoh and co-workers (Itoh et al., Chem. Commun. 1997: 677-678) reported that the hydrolysis of ethyl(2,4-dinitrophenyl)phosphate by the copper(II) complex of cis,cis-1,3,5-triaminocyclohexane (tach) was significantly greater than that for several other tramino ligands. Until recently, the availability of tach and derivatives thereof was limited by the synthetic procedure.
In view of the above, it is an object of the present invention to provide new synthetic metallonucleases, in particular transition metal complexes of novel derivatives of tach. The novel derivatives of tach enable the formation of complexes with many transition metals and the resultant metallonucleases cleave activated phosphate diesters at an unprecedented rate and demonstrate concentration-dependent cytotoxicity. It is a related object of the present invention to provide a method of synthesizing tach. Such a synthesis route does not suffer from the disadvantages of complexity and low yield associated with currently available methods of synthesis, being less complicated and providing tach in high yield. It is another object of the present invention to provide conjugates of the transition metal complexes of novel derivatives of tach. It is yet another object of the present invention to provide methods of using such complexes and conjugates thereof. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the detailed description provided herein.
The present invention provides a transition metal complex of N,Nxe2x80x2,Nxe2x80x3-trialkyl-cis,cis-1,3,5-triaminocyclohexane. Preferably, the transition metal is copper, in particular copper II (Cu (II)). The trialkyl preferably comprises C1-C6 alkyl groups, which may be the same or different. Preferably, the trialkyl is trimethyl or triethyl, with trimethyl being especially preferred. The transition metal can be radioactive, such as a positron emitter, e.g., Cu64, or a xcex2-emitter, e.g, Cu67.
The transition metal complex of N,Nxe2x80x2,Nxe2x80x3-trialkyl-cis,cis-1,3,5-triaminocyclohexane can be conjugated to a targeting agent. Preferably, the targeting agent is an immunological agent, a protein, a polypeptide, a peptide, a nucleic acid or a steroid.
Also provided by the present invention is a composition comprising a transition metal complex of N,Nxe2x80x2,Nxe2x80x3-trialkyl-cis,cis-1,3,5-triaminocyclohexane, or a conjugate thereof, and a carrier therefor.
In addition to the above, the present invention provides a method of synthesizing N,Nxe2x80x2,Nxe2x80x3-trialkyl-cis,cis-1,3,5-triaminocyclohexane. The method comprises derivatizing 1,3,5-cis,cis-triaminocyclohexane to a tris-sulfonamide, removing the sulfonamide proton with a base to generate a tris-anion, quenching the tris-anion with an alkylating agent, and cleaving the sulfonamide group with an acid to generate N,Nxe2x80x2,Nxe2x80x3-trialkyl-cis,cis-1,3,5-triaminocyclohexane as a protonated salt. The method can further comprise adding an equimolar amount of CuX2 in water to the N,Nxe2x80x2,Nxe2x80x3-trialkyl-cis,cis-1,3,5-triaminocyclohexane, neutralizing the resulting solution with base solution (to form a deep-blue solution), and removing the hydroxides that form by filtration to yield a solution of the Cu (II) complex.
Further provided by the present invention is a method of cleaving a biological molecule. The method comprises contacting the biological molecule with an above-described complex, which cleaves the biological molecule. Preferably, the biological molecule is in vivo and the cleavage of the biological molecule has a therapeutic effect. Also preferably, the complex is targeted to an abnormally proliferating cell that comprises the biological molecule, such as a cancerous cell and the therapeutic effect is the treatment of cancer.
Still further provided by the present invention is a method of inhibiting expression of a nucleic acid. The method comprises contacting a nucleic acid with an above-described transition metal complex conjugated to an antisense nucleic acid specific for the nucleic acid, whereupon the antisense nucleic acid binds to the nucleic acid, thereby inhibiting expression of the nucleic acid.
In another embodiment, the present invention provides a method of radiation therapy. The method comprises administering to an animal in need of radiation therapy a therapeutically effective amount of an above-described radioactive complex, which is (i) conjugated to a targeting agent that binds to a molecule on the surface of a cell to be treated with radiation or (ii) encapsulated in a liposome comprising on its surface the targeting agent.
In yet another embodiment, a method of imaging is provided. The method comprises (i) administering to an animal an imaging effective amount of an above-described radioactive complex, which is either conjugated to a targeting agent that binds to a molecule on the surface of a cell to be imaged or encapsulated in a liposome comprising on its surface the targeting agent, and (ii) imaging the complex. Preferably, the targeting agent is an immunological agent.
In still yet another embodiment, the present invention provides a method of tracing a compound in an animal. The method comprises administering to an animal a mixture of the compound and an above-described complex and tracing the location of the complex in the animal.